3-methoxyoestra-1,3,5(10)-trien-17-one

Endpoint summary

Description of key information

local lymph node assay: not skin sensitizing (van Otterdijk, 2011); read-across from Estrone

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. The formulations were stirtred with magnetic stirrer immediately prior to dosing.

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation:
- Housing: group housed
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d
A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0ºC (actual range: 17.6 - 21.6ºC),
- Humidity (%): 40-70% (actual range: 21 – 64%)
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

Pre-Screen test: 25% and 50%
Main test: 10%, 25% and 50%

No. of animals per dose:

Pre-Screen test: 2
Main Test: 5

Details on study design:

PRE-SCREEN TESTS:
- tested at 25% and 50% concentration
- Compound solubility:
- Irritation: Slight irritation along with white test substance remnants (which did not hamper scoring) were observed on both ears of all animals treated at a 25 and 50% concentration between Days 1 and 3
- Systemic toxicity: no
- Ear thickness measurements: no


MAIN STUDY

- Criteria used to consider a positive response:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
On the induction days 1,2 and 3 the dorsal surface of both ears was topically treated (25 µl/ear) with the test substance concentration.

On day 6 the draining (auricular) lymph node of each ear was excised after injection of 3H-methyl thymidine via tail vein to the mice 5 h before and tissue was processed for radioactivity measurements.

Additionally, clinical signs (daily), body weights (day 1 and 6) and signs of irritation or other local effects were observed.

Positive control substance(s):

other:

Positive control results:

reliability check:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 2.2 and 3.7 respectively. An EC3 value of 18.0% was calculated using linear interpolation.
The calculated EC3 value was found to be in the expected range of 2 and 20%. The results of 6 monthly HCA reliability checks of the recent years were 13.8, 13.9, 16.0, 11.9, 16.9 and 10.7%.

Parameter:

SI

Value:

1.6

Variability:

+- 0.4

Test group / Remarks:

5 females/10 %-group

Key result

Parameter:

SI

Value:

1.7

Variability:

+- 0.5

Test group / Remarks:

5 females/25 %-group

Parameter:

SI

Value:

0.9

Variability:

+- 0.2

Test group / Remarks:

5 females/50 %-group

Parameter:

SI

Value:

1

Variability:

+- 0.2

Test group / Remarks:

vehicle control group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

449

Test group / Remarks:

10 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

470

Test group / Remarks:

25 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

268

Test group / Remarks:

50 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

283

Test group / Remarks:

vehicle control group

No mortalities occurred, no symptoms of systemic toxicity, no changes in body weights and body weight gain were observed.

A slight irritation (grade 1) of the ears was seen in all animals treated with 25 and 50%, but this was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema were observed.

All auricular lymph nodes were considered normal in size, no macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:

GHS criteria not met

Conclusions:

not skin sensitizing

Executive summary:

To determine the skin-sensitizing properties of estrone the mouse local lymph node assay was performed on female CBA/J mice (5/group) according to OECD guideline 429. The main study was conducted with the following test substance concentrations: 0 (vehicle control), 10%, 25% and 50% formulated in propylene glycol.

All animals treated with 25 and 50% showed slight irritation of the ears, but this had no toxicological significant effect on the activity of the lymph nodes. White test substance remnants were present on the ears of all animals at 10, 25 and 50%, which did not hamper scoring of the skin irritation reactions.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10,

25 and 50% were 449, 470 and 268 DPM respectively. The mean DPM/animal value for the vehicle

control group was 283 DPM.The mean DPM/animal values were 283 (vehicle control), 449 (10%), 470 (25%) and 268 (50%). The SI values calculated were 1.6 (10%), 1.7 (25%) and 0.9 (50%). Since there was no indication that the test substance elicits an SI above or equal to 3 when tested up to 50%, estrone was considered not to be a skin sensitizer.

A regularly performed reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

HYPOTHESIS FOR THE ANALOGUE APPROACH

Regarding human health, toxicity studies for estrone 3-methyl ether (estrone-3-methylether) and its structural relative estrone are available according to REACH requirements of Annex VII (1-10 t/a). The underlying mechanism linking these substances is the effect they exert on the estrogen receptor, although the potency is quantitatively somewhat different.
Estrone shows a pharmacological profile (comprising the binding to different hormone receptors) similar to estrone-3-methylether which suggests the comparison of the biological activity regarding human health toxicology of both substances after becoming systemically available and to justify the use of estrone as a source for read-across to estrone-3-methylether as target. Regarding the underlying biological mechanism of acting on the estrogen receptor estrone is more potent than estrone-3-methylether and can therefore be considered as a worst-case scenario.

The source substance estrone and the target substance estrone-3-methylether are structurally similar, moderately lipophilic substances, which are absorbed after administration and differently (bio)transformed but leading to the same final metabolites. The exposure to source and target substance causes the same type of effects through a common mechanism: induction of pharmacologic effects via the estrogen receptor.
The strength of effects of the source substance is predicted to be the worst-case scenario for the effects of the target substance for the property under consideration. The common metabolites (2- or 4-Hydroxyestrone-3-methyl ether) are assumed to be less biological active compared to estrone.
It is further assumed that the pharmacological effect dominates the biological activity of estrone-3-methylether and toxicological effects due to off-target binding are not anticipated below this pharmacological effect level.
Whereas estrone is an endogenous steroid hormone that can be (reversely) converted into estradiol and serves mainly as a precursor or metabolic intermediate of estradiol, estrone-3-methylether is a synthetic steroid hormone which is metabolized only into one direction but not reverse.
The read-across hypothesis is supported by the finding that CYP450-mediated metabolism of estrone and estrone-3-methylether was similar in rat liver microsomes in vitro.
In summary, it can be concluded that estrone and estrone-3-methylether exert very similar comparable biological effects in vivo via the estrogen receptor.

A detailed justification for read-across is attached on iuclid section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Value:

1.6

Variability:

+- 0.4

Test group / Remarks:

5 females/10 %-group

Parameter:

SI

Value:

1.7

Variability:

+- 0.5

Test group / Remarks:

5 females/25 %-group

Parameter:

SI

Value:

0.9

Variability:

+- 0.2

Test group / Remarks:

5 females/50 %-group

Parameter:

SI

Value:

1

Variability:

+- 0.2

Test group / Remarks:

vehicle control group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

449

Test group / Remarks:

10 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

470

Test group / Remarks:

25 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

268

Test group / Remarks:

50 %-group

Parameter:

other: mean disintegrations per minute (DPM)/animal

Value:

283

Test group / Remarks:

vehicle control group

Conclusions:

not skin sensitizing

Executive summary:

No data on sensitising properties is available for the target substance estrone-methylether. Results of a study conducted with estrone are regarded as representative based on close structural similarity as further detailed in the Justification for read-across attached to Iuclid section 13.

 

To determine the skin-sensitizing properties of estrone the mouse local lymph node assay was performed on female CBA/J mice (5/group) according to OECD guideline 429. The main study was conducted with the following test substance concentrations: 0 (vehicle control), 10%, 25% and 50% formulated in propylene glycol.

All animals treated with 25 and 50% showed slight irritation of the ears, but this had no toxicological significant effect on the activity of the lymph nodes. White test substance remnants were present on the ears of all animals at 10, 25 and 50%, which did not hamper scoring of the skin irritation reactions.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 449, 470 and 268 DPM respectively. The mean DPM/animal value for the vehicle control group was 283 DPM.The mean DPM/animal values were 283 (vehicle control), 449 (10%), 470 (25%) and 268 (50%). The SI values calculated were 1.6 (10%), 1.7 (25%) and 0.9 (50%). Since there was no indication that the test substance elicits an SI above or equal to 3 when tested up to 50%, estrone was considered not to be a skin sensitizer.

A regularly performed reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity.

Based on read-across, estrone-methylether is not a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

With respect to skin sensitizing effects, no experimental study data for estrone-3-methylether is available. Read-across is applied using experimental in vivo data in mice (Local lymph node assay; LLNA according to OECD TG 429) with estrone as source. Substance purity of estrone in this guideline GLP study was >95% and reliability of the study report is allocated to Klimisch score 1.

 

To determine the skin-sensitizing properties of estrone the mouse local lymph node assay was performed on female CBA/J mice (5/group) according to OECD guideline 429. The main study was conducted with the following test substance concentrations: 0 (vehicle control), 10%, 25% and 50% formulated in propylene glycol.

All animals treated with 25 and 50% showed slight irritation of the ears, but this had no toxicological significant effect on the activity of the lymph nodes. White test substance remnants were present on the ears of all animals at 10, 25 and 50%, which did not hamper scoring of the skin irritation reactions.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10,

25 and 50% were 449, 470 and 268 DPM respectively. The mean DPM/animal value for the vehicle

control group was 283 DPM. The mean DPM/animal values were 283 (vehicle control), 449 (10%), 470 (25%) and 268 (50%). The SI values calculated were 1.6 (10%), 1.7 (25%) and 0.9 (50%). Since there was no indication that the test substance elicits an SI above or equal to 3 when tested up to 50%, estrone was considered not to be a skin sensitizer.

A regularly performed reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at the laboratory is an appropriate model for testing for contact hypersensitivity (van Otterdijk, 2011).

 

No sensitization potential for estrone was demonstrated in this study, i.e. no relevant increase in DPM and SI-values compared to controls up to the highest concentration used (50%). Slight and reversible local irritation of the skin after topical application of estrone to the murine ears in the 25% and 50% concentration groups did not result in any lymph node findings and was therefore not regarded as adverse effect related to the outcome of this study. Dermal absorption based on enhanced lipophilicity and logPow values of estrone and estrone-3-methylether is assumed to be similar for both substances therefore justifying a read-across approach for this particular endpoint. Further biological activity of both substances is due to the activation of the estrogen receptor with estrone (source) representing a worst-case scenario for estrone-3-methylether (target).

 

The experimental result for estrone is supported by the safe dermal application of other estrogens (in particular the more potent estradiol) in several clinical studies without adverse or sensitization findings (Rzepecki, 2019). For example, a 6-month topical treatment of 59 preclimateric women with skin-aging symptoms with 0.01% estradiol and 0.3% estriol compounds was was tolerated well without skin reactions (Schmidt, 1996). In general, the systemic absorption of local or topical estrogen therapies is thought to be quite low (Klinger, 2002). Although a clinical case study published in 1995 demonstrated increased sensitivity in 6 of 7 women after intradermal application (subepidermal injection of 0.1 ml) of estrogens including estrone (Shelley, 1995) this finding can be regarded as a very rare event.

 

In addition, an in silico assessment of skin sensitization (QSAR Toolbox 4.3.1) for estrone and estrone-3-methylether was carried out. For estrone the QSAR Toolbox reveals ‘Protein binding alerts for skin sensitisation by OASIS’ (identical with estrone-3-methylether) - but not ‘Protein binding alerts for skin sensitisation according to GHS’. Both profilers showed alerts for estrone-3-methylether. All alerts are based on the reactivity of the ketone group (addition to the carbon-hetero double bond) and no other skin sensitization profiler showed any alert. The negative experimental LLNA data for estrone indicate that under in vivo conditions no skin sensitization occurs in contrast to the positive QSAR alert.

The presence of a methoxy-group or hydroxyl-group in the molecule is regarded as not relevant for skin sensitization as these groups are not able for protein binding as a basic requirement for skin sensitization.

Further, the anticipated metabolism of estrone-3-methylether by enzymatic CYP450 degradation in the epidermis of the skin (refer to 4.2) resulting in the endogenous 2- or 4-Hydroxyestrone-3-methyl ether will not increase immunogenicity of the hapten. Consequently, estrone-3-methylether (CAS 1624-62-0) is concluded as not sensitizing.

 

Based on the LLNA data for estrone as read-across source and the comparison to the in silico prediction results for both substances no skin sensitizing potential can be concluded for estrone-3-methylether epicutaneously and therefore classification (Reg. (EC) No. 1272/2008) is not warranted.

 

QSAR predictions and LLNA sensitization of estrone for read-across to estrone-3-methylether

	Estrone	Estrone-3-methylether
CAS No.	53-16-7	1624-62-0
ZK	5019	5512
QSAR prediction (Toolbox 4.3.1); Report Schlecker, 2019
Profilers with protein binding alerts for skin sensitization	1 (OASIS)	2 (OASIS & GHS)
Alerts	Nucleophilic addition/ Addition to Carbon-hetero double bonds/ Ketones	·         Nucleophilic addition/ Addition to Carbon-hetero double bonds/ Ketones ·         Skin sensitization Category 1B >> Ketones
LLNA study
Study no./ Report no.	NOTOX 495755 (Otterdijk, 2011)	No data     Read-across
GLP/ OECD TG/ deviations	GLP, OECD 429; no deviations   Regular reliability check with α-hexylcinnamic-aldehyde
Species; animals/ group	Mice, female n=5/ group (main study)
Compound purity/ formulation	·         Batch EFB465K1A ·         Purity >95% ·         Vehicle: propylene glycol ·         freshly prepared, homogeneity visually confirmed
Doses tested	10%, 25% and 50% w/w, dose selection based on pre-test
Route/ schedule	Topical treatment of ears on d1, d2 and d3
Parameters assessed	Skin reactions, auricular lymph node assessment, BW in comparison to vehicle, radioactivity measurements of activated LN cells, systemic toxicity
Results	no skin sensitization
Reliability	1

 

 

References:

Klinger W. et al., Toxicology Letters 128 (2002) 129–144

Rzepecki et al. Int J Womens Dermatol. 2019 Jun; 5(2): 85–90

Schmidt et al. 1996; Int J Dermatol Vol 35, No. 9, 669

Shelley et al. 1995, Journal of the American Academy of Dermatology, Volume 32, Issue 1, January 1995, Pages 25-31

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data for surrogate no classification for skin sensitisation according to Regulation (EC) No. 1272/2008 (CLP) is required for estrone methyl ether.